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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chemical investigations of the human growth hormone (HGH) molecule were briefly reviewed for th period between 1974 and 1981. These include chemical modification, selective enzymic cleavage, the 20K HGH, synthetic peptide fragments, complementation of the natural NH2-terminal 134-amino acid fragment with natural or synthetic COOH-terminal fragments of various chain lengths, covalent reconstitution of two contiguous fragments of HGH with thrombin and bacterially synthesized methionyl-HGH.
Mol Cell Biochem 1982 Jul 07
PMID:Human growth hormone: 1974-1981. 705 Jun 55

Free energy calculations were carried out on a series of exosite-binding inhibitors of thrombin. These inhibitors are based on the C-terminal fragment of hirudin and have the sequence Phe-Glu-Glu-IleH59-Pro-Glu-Glu-Tyr- Leu, where the superscript over Ile indicates its relative position in the natural sequence of hirudin. In this study, the effect of replacing IleH59 with ten other non-polar amino acids was examined. Three preferred interaction sites for methyl/methylene groups for the various XaaH59 side-chains in the complex were identified from conformational search calculations. The corresponding thermodynamic changes were determined using a combination of systematic search and energy minimization in a manner that locates the local minima in the system and in the process simultaneously builds up the partition function. The free energy, internal energy and entropic contributions are readily calculated from the partition function. Very good agreement in the resulting relative binding free energies was obtained between theory and experiment. The calculations allowed us to dissect out the enthalpic, entropic and solvation contributions to delta delta G. The contribution from desolvation was found to be relatively weak. The binding of these non-polar side-chains to thrombin is found to be driven mainly by favorable protein-ligand interactions rather than by the desire for non-polar groups to be desolvated. We also find that the configurational entropy contributes about 0.48 kcal/mol (0.81 kappa T) in average for each torsional angle "frozen" in binding.
J Mol Biol 1995 Oct 27
PMID:Calculation of relative binding free energies and configurational entropies: a structural and thermodynamic analysis of the nature of non-polar binding of thrombin inhibitors based on hirudin55-65. 747 28

Temperature dependent studies of the interaction of the clotting enzyme thrombin with the potent natural inhibitor hirudin reveal a large negative heat capacity change of -1.7(+/- 0.2) kcal/mol per K associated with the formation of the thrombin-hirudin complex, independent of the allosteric state of the enzyme. Binding of N-terminal fragments of hirudin (hir1-49 and hir1-43) is characterized by heat capacity changes of -1.2(+/- 0.1) and -0.9(+/- 0.1) kcal/mol per K, respectively. The magnitude of these heat capacity changes is unprecedented for protease-inhibitor interactions. A thermodynamic analysis based on observed heat capacity and entropy changes predicts that binding is accompanied by substantial coupled folding transitions in both hirudin and thrombin. In the absence of a structure of free thrombin, analysis of differences in the predicted number of residues which fold upon binding hirudin and its fragments leads to the following structural model: three surface loops in thrombin (W60d, W148 and fibrinogen binding loops) are disordered in the free state and fold upon formation of the thrombin-hirudin complex. Molecular dynamics simulations, run over a time scale of 5 ps, are consistent with the hypothesis of large scale coupled folding transitions in both hirudin and thrombin upon formation of the complex. Comparison of the thermodynamics for the interaction of hirudin with the slow and fast forms of thrombin allows dissection of the coupling free energy for allosteric switching. The coupling free energy for the slow-->fast transition increases linearly, in absolute value, with temperature. The coupling enthalpy and entropy terms for hirudin were found to be delta Hoc = 12(+/- 1) kcal/mol and delta Soc = 47(+/- 4) cal/mol per K. Preferential interaction with the fast form is therefore due to the balance of two opposite forces, both quite large in magnitude. The contribution of enthalpic effects opposes the slow-->fast transition and stabilizes binding to the slow form. The contribution of entropic effects favors the slow-->fast transition and stabilizes binding to the fast form. In the physiological temperature range the entropic effects prevail and result in preferential binding of hirudin to the fast form. The region of thrombin recognizing the N-terminal domain of hirudin contains most of the residues that are energetically linked to the slow-->fast transition. This region is part of the "allosteric core" of thrombin and includes the W60d loop, shaping the specificity site S2, and the Na+ binding loop connecting the last two beta-strands of the B chain.
J Mol Biol 1995 Nov 10
PMID:Thermodynamic investigation of hirudin binding to the slow and fast forms of thrombin: evidence for folding transitions in the inhibitor and protease coupled to binding. 747 52

The vascular angiotensin II (ANG II) receptor (AT1) is a central component of the renin-angiotensin system; thus, regulation of its expression is likely to be important in cardiovascular responsiveness. We demonstrate that ANG II down-regulates its receptor in rat aortic vascular smooth muscle cells. Incubation for 4 hr with 100 nM ANG II decreased AT1 mRNA and protein by 70% and 35%, respectively. This homologous down-regulation was concentration and time dependent and was blocked by the AT1 antagonist losartan. It did not appear to be mediated by protein kinase C or other protein kinases but was dependent on the sustained signaling pathway sensitive to phenylarsine oxide. Heterologous down-regulation was observed with the agonists alpha-thrombin and ATP and the cAMP-increasing agent forskolin. ANG II inhibited transcription by 50% and destabilized the AT1 mRNA. Down-regulation of AT1 mRNA was blocked by transcription and translation inhibitors, suggesting that it required expression of a protein factor or factors. These results indicate that ANG II down-regulates its vascular receptor by both transcriptional and post-transcriptional mechanisms. Homologous and heterologous down-regulation of the AT1 receptor may participate in the coordinated physiological adaptation of vascular tone to vasoactive hormones.
Mol Pharmacol 1995 Oct
PMID:Angiotensin II down-regulates the vascular smooth muscle AT1 receptor by transcriptional and post-transcriptional mechanisms: evidence for homologous and heterologous regulation. 747 84

In addition to its procoagulant properties, the serine protease thrombin increases endothelial permeability, stimulates granulocyte adherence, and serves as a fibroblast mitogen. We demonstrate that thrombin is mitogenic for human lung fibroblasts in vitro. The mitogenic effect of thrombin is associated with an increase in the expression of the ligand PDGF-AA and up-regulation of PDGF alpha-receptor. Since scleroderma (systemic sclerosis; SSc) is characterized by widespread microvascular injury and is frequently complicated by pulmonary fibrosis, we sought to determine the level of thrombin activity in bronchoalveolar lavage (BAL) fluid from SSc patients and normal controls. We report a significantly higher level of thrombin activity in BAL fluid from SSc patients compared with normal controls (P < 0.001). Taken together, the high levels of thrombin in BAL fluid and its demonstrated mitogenicity for lung fibroblasts suggest an important role for thrombin in the pathogenesis of SSc and perhaps other fibrotic lung diseases.
Am J Respir Cell Mol Biol 1994 Apr
PMID:Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor. 751 Sep 86

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.
Mol Cell Biol 1994 Apr
PMID:Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes. 751 Dec 5

The cDNA sequences of chicken and hagfish prothrombin have been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringle domains, and a two-chain protease domain. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues). Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the propeptide-Gla domain are the regions most constrained for the common function(s) of vertebrate prothrombins.
J Mol Evol 1994 Feb
PMID:Evolution of prothrombin: isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin. 751 65

Intra-alveolar fibrin deposition is a cardinal feature of neonatal respiratory distress syndrome and likely contributes to short-term and long-term morbidity. Previous studies have shown that fetal distal lung epithelial cell (FDLE) surfaces express procoagulant activity when incubated with adult plasma and may therefore provide one mechanism by which fibrin is generated. However, plasma concentrations of prothrombin and thrombin inhibitors differ significantly at birth and during the first weeks of life compared with adult values. Therefore, we measured thrombin-generating capacity and inhibitor complex formation in cord and adult plasma incubated in the presence of FDLE. Although starting cord plasma concentrations of prothrombin were 43% of adult values, the amount of thrombin generated was decreased by only 21%. When cord plasma concentrations of prothrombin were selectively increased to adult values, the amount of thrombin generated surpassed adult plasma by 89%. The latter observations suggested that thrombin inhibition was impaired in cord plasma compared with adult plasma and supplementation of cord plasma with antithrombin III (ATIII) as well as prothrombin returned thrombin generation to adult levels. However, the percentage of thrombin complexed to inhibitors (59%) at the completion of the experiments was similar in cord, cord plus prothrombin, cord plus prothrombin plus ATIII, and adult plasmas. Although a higher proportion of thrombin was inhibited by alpha 2-macroglobulin (alpha 2M) in cord plasma and cord plasma plus prothrombin, this did not compensate for the decreased amount of thrombin inhibited by ATIII. When cord plasma was supplemented with ATIII as well as prothrombin, the proportions of thrombin complexed by the different inhibitors were similar to those of adult plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1994 Jul
PMID:Thrombin inhibition by fetal distal lung epithelium is different in fetal and adult plasma. 751 42

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.
Mol Biol Cell 1994 May
PMID:Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain. 752 56

Protein tyrosine kinases (PTKs) of the src family are thought to play an important role in platelet signal transduction, but little is known about the targets of these enzymes in platelets. We determined that exposure of human platelets to pervanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in the activity of phospholipase D (PLD), an enzyme that might be involved in signaling events leading to aggregation or secretion. To further investigate whether tyrosine phosphorylation is a step in the pathway of activation of PLD in response to thrombin, we tested the effects of a series of PTK inhibitors on the activity of platelet PLD. PLD was activated in response to 0.3 U/ml thrombin, and this activation was reduced by several of the PTK inhibitors, especially genistein, methyl 2,5-dihydroxycinnamate (MDHC), ST271, and the tyrphostins A25 and A47. In saponin-permeabilized platelets, we observed a marked inhibition of GTP-gamma S-stimulated PLD by many of the PTK inhibitors, consistent with the possibility that PTKs are involved in the regulation of PLD activity by a G-protein or small GTP-binding protein. MDHC did not affect PLD activity in permeabilized cells, which suggests that this compound might inhibit PLD in intact platelets via another pathway. The inhibitors were also tested for their effects on the phosphorylation of a peptide substrate of src-family PTKs in a platelet membrane preparation and in permeabilized platelets. Several of the compounds partially inhibited peptide phosphorylation in the membrane preparation and in permeabilized platelets, most notably ST271, ST638, and tyrphostin A25.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Mol Biol (Noisy-le-grand) 1994 Jul
PMID:Inhibition of phospholipase D of human platelets by protein tyrosine kinase inhibitors. 752 16


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